Human tumor necrosis factor-α (hereinafter, referred to as “hTNF-α”) is a homotrimer consisting of three 17 kDa protein subunits (Eck M. J. et al., JBC, 267: 2119-2122, 1992; Smith R. A. et al., JBC, 262: 6951-6954, 1987). hTNF-α is an inflammatory cytokine secreted from macrophages and monocytes, and functions as a signal transmitter in several cellular reactions such as necrosis and apoptosis (Beyaert R. et al., FEBS Lett., 340: 9-16, 1994). hTNF-α causes a pro-inflammatory action leading to tissue destruction, such as breakdown of the cartilage and bone (Saklatvala, Nature, 322: 547-549, 1986), induction of an adhesion molecules, induction of procoagulation activity in vascular endothelial cells (Pober J S et al., J. Immunol., 136; 1680-1687, 1986), and increase in the adherence of neutrophils and lymphocytes (Pober et al., J. Immunol. 138: 3319-3324, 1987). In addition, it has been known that hTNF-α plays an important role in a defense mechanism against infectious disease and tumor (Fiers W., FEBS Lett., 285: 199-212, 1991).
hTNF-α is involved in inflammatory diseases, autoimmune diseases, bacterial infections, cancers and degenerative diseases. Among these diseases, hTNF-α has been regarded as a useful target protein for a specific physiological treatment of rheumatoid arthritis, Crohn's disease, psoriatic arthritis, and ankylosing spondylitis.
Meanwhile, it has been also suggested to use a hTNF-α inhibitor for the purpose of treating rheumatoid arthritis. It has been reported that hTNF-α is overexpressed in the synovial cells isolated from the early-stage rheumatoid joint (Buchan G. et al., Clin. Exp. Immunol., 73: 449-455, 1988), and cytokines relating to rheumatoid arthritis lesions are decreased when the above synovial cells are treated with an anti-hTNF-α monoclonal antibody (Butler D. M. et al., Eur. Cytokine Netw., 6: 225-230, 1995). Further, it has been found that an anti-hTNF-α antibody or a recombinant soluble hTNF-α receptor suppresses inflammation and destruction of a joint in a collagen induced mouse arthritis model (Piguet P. F. et al., Immunology, 77: 510-514, 1992; Wooley P. H. et al., J. Immunol., 151: 6602-6607, 1993; Williams R. O. et al., Immunology, 84: 433-439, 1995). Moreover, it has been observed that inflammatory arthritis is induced in a transgenic mouse overexpressing hTNF-α (Keffer J. et al., EMBO J., 10: 4025-4031, 1991).
These results indicate that hTNF-α plays an important role as a direct or indirect regulator controlling inflammatory cytokines in rheumatoid athritis. Accordingly, there has been a need to develop a monoclonal antibody having high selectivity and reactivity to hTNF-α for the purpose of treating rheumatoid arthritis.
Generally, an antibody having high selectivity and reactivity to a specific antigen is prepared through immunization of a mouse with the antigen (Kohler G. & Milstein C., Nature, 256; 495-497, 1975). A mouse monoclonal antibody is advantageous in that it is easy to prepare the antibody and to select an antibody having a high reactivity. However, it also has a problem that an anti-mouse antibody is formed in a human body when it is administered for a long time (Dimaggio J. J., et al., Cancer Chemother. Biol. Response Modif., 11: 177-203, 1990).
To overcome the undesirable properties of mouse monoclonal antibodies, a humanized antibody has been developed by replacing the framework regions except for the antigen-binding site with those of a human antibody. As a method for preparing such humanized antibody, a CDR (complementarity determining region) grafting method is currently employed, wherein only the CDRs of a mouse antibody are grafted to a human antibody. The humanized antibody prepared by CDR grafting has an advantage of reducing in vivo immune responses (Riechmann et al., Nature, 332: 323, 1988; Nakatani et al., Protein Engineering, 7: 435, 1994), however, it often loses the high selectivity and reactivity of the original mouse antibody (Carter P., et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289, 1992).
The present inventors have endeavored to overcome such problems of the conventional humanized antibody, and developed a novel humanized antibody specifically binding to hTNF-α, which shows an antigen binding affinity similar to that of the original mouse monoclonal antibody and minimized immunogenicity.